Mechanisms of Diacylglycerol Signaling Through C1 Domain Proteins Diacylglycerol (DAG) is a paradigmatic lipid second messenger in metazoan cell signaling, the first to be discovered. The protein kinase C family is the best known target for DAG signals, but other proteins, such as the chimaerins, have also come to the fore as important receptors. PKCs and other DAG receptors are therapeutic targets for cancer, diabetes, and immunosuppression, among others. PKCs and other DAG receptors bind DAG through their C1 domains. This binding event triggers both membrane translocation and allosteric activation through conformational changes. This project aims to characterize these activation mechanisms at atomic-level detail. In addition to the structure of the phorbol ester:PKC delta C1B domain complex , past work in this project has yielded the first and only structure of a full-length C1 domain protein, that of beta2 chimaerin. The approaches used are 1) the crystallization of full-length C1-domain containing proteins;2) analysis of activation dynamics using molecular simulations;and 3) spectroscopic analysis of activation kinetics and the conformation of active, membrane-associated states inaccessible to crystallization. Diacylglycerol is a central mediator of downstream signaling by a host of hormones coupled through Gq and phospholipase Cbeta, growth factors coupled to tyrosine-kinase linked receptors and phospholipase Cgamma, and many other extra- and intracellular stimuli. The protein kinase C (PKC) isozyme family has historically been the most intensively studied class of targets for diacylglycerol signaling. PKC isoforms are under active investigation as therapeutic targets for cancer and retinopathy in diabetes, and as targets for immunosuppression. In response to diacylglycerol, conventional and novel PKC isozymes, and the protein kinase D isozymes, translocate to membranes, where they phosphorylate Ser and Thr residues in diverse proteins. The diacylglycerol-responsive PKC isozymes are also activated by phorbol esters, which acts as a potent agonist by binding to the same site as diacylglycerol. These PKC isozymes are activated by diacylglycerol and phorbol esters by virtue of their direct binding to motifs known as protein kinase C homology-1 (C1) domains. C1 domains are compact zinc fingers of 50-51 amino acids. Their structure comprises two small b sheets and a single helix folded around two Zn 2+ ions. The two strands in the smaller b sheet are pulled apart, due to a break in their hydrogen bonding induced by a conserved Pro residue. Phorbol ester binds stereospecifically in the groove formed where the two strands pull away from each other, first described by structural studies from this laboratory. The stereospecific phorbol ester binding site is surrounded on three side by bulky hydrophobic side chains, which form a hydrophobic wall around the phorbol ester binding site. Distal to the phorbol ester-binding site, Arg, Lys, and His residues form a basic belt on the surface of the C1 domain. The hydrophobic wall inserts in the hydrocarbon core of phospholipid bilayers upon binding, while the basic belt interacts with acidic phospholipid headgroup. The high affinity of C1 domain/phorbol ester interactions in the presence of bilayers or micelles is due to the synergism between the stereospecific phorbol ester binding and nonspecific binding of acidic phospholipids to the basic and hydrophobic exterior surface of the C1 domain. Translocation and activation of C1 domain proteins requires the interplay of specific and nonspecific activators: both diacylglycerol or phorbol ester and bulk phospholipid. Efforts in FY2009 concentrated on the crystallization and small angle x-ray scattering analysis of full-length PKC isozymes. We continue to make incremental progress towards this challenging long-term goal.